Complete organelle genomes of the threatened aquatic species Scheuchzeria palustris (Scheuchzeriaceae): Insights into adaptation and phylogenomic placement

Abstract Scheuchzeria palustris, the only species in the Scheuchzeriaceae family, plays a crucial role in methane production and transportation, influencing the global carbon cycle and maintaining ecosystem stability. However, it is now threatened by human activities and global warming. In this study, we generated new organelle genomes for S. palustris, with the plastome (pt) measuring 158,573 bp and the mitogenome (mt) measuring 420,724 bp. We predicted 296 RNA editing sites in mt protein‐coding genes (PCGs) and 142 in pt‐PCGs. Notably, abundant RNA editing sites in pt‐PCGs likely originated from horizontal gene transfer between the plastome and mitogenome. Additionally, we identified positive selection signals in four mt‐PCGs (atp4, ccmB, nad3, and sdh4) and one pt‐PCG (rps7), which may contribute to the adaptation of S. palustris to low‐temperature and high‐altitude environments. Furthermore, we identified 35 mitochondrial plastid DNA (MTPT) segments totaling 58,479 bp, attributed to dispersed repeats near most MTPT. Phylogenetic trees reconstructed from mt‐ and pt‐PCGs showed topologies consistent with the APG IV system. However, the conflicting position of S. palustris can be explained by significant differences in the substitution rates of its mt‐ and pt‐PCGs (p < .001). In conclusion, our study provides vital genomic resources to support future conservation efforts and explores the adaptation mechanisms of S. palustris.

to a sharp decline in its wild populations and it has been classified as a threatened species in many European countries (Smith et al., 2021).In China, S. palustris is sporadically found in the alpine or sub-alpine swamps of the Changbai Mountain region, near the southernmost boundary of its range and extremely vulnerable to climate warming, which leads to its inclusion as a Grade II protected plant in China (Lu et al., 2021).
Previous studies on S. palustris have primarily focused on its ecology (Moore, 1955), floral development (Volkova et al., 2016), and phylogeny (Li et al., 2023;Ross et al., 2016).However, there is still a lack of basic genetic resources for this unique aquatic plant, which limits further research and conservation efforts for this threatened species.
These organelles play key roles in cellular processes in plants, particularly in energy metabolism.Chloroplasts, the primary sites of photosynthesis in plant cells, convert solar energy into carbohydrates and oxygen, while mitochondria serve as the cell's energy center, supplying the energy required for numerous cellular processes in plants (Lai et al., 2022;Liberatore et al., 2016;Tang et al., 2020).
The number of documented plastomes significantly surpasses that of mitogenomes due to the relatively simpler structure (ca.120-130 genes) and smaller size (ca.120-170kb) of plastomes in angiosperms, which typically exhibit a conserved four-part architecture containing inverted repeats (IRs) and single-copy regions (Henry, 2005;Jansen & Ruhlman, 2012).In contrast, plant mitogenomes are generally larger and more complex, characterized by varying genome sizes (ca.100-10,000 kb), diverse conformations, low gene densities, and an abundance of repetitive sequences and RNA editing events (Jansen & Ruhlman, 2012), making the conformation of plant mitogenome challenging (Gualberto et al., 2014).Moreover, horizontal gene transfer (HGT) of DNA from plastome to mitogenome in angiosperms is a common phenomenon, which might affect organelle gene function and expression (Kim et al., 2023;Straub et al., 2013), thereby influencing adaptation to stress resistance (i.e., cold and high altitude).
Due to the ease of assembling plastomes, nearly 10,000 plant plastomes have been reported and widely applied to resolve phylogeny at different taxonomic levels (Wang, Kan, et al., 2024;Wang, Liu, et al., 2024).Our previous research reported the plastome of S. palustris and explored the dynamic history of plastome structure across Alismatidae (Li et al., 2023).However, research on the mitogenome of S. palustris remains limited.With the recent advancements in sequencing technologies and assembly methods, obtaining plant mitogenomes has become feasible (Bi, Shen, et al., 2024;Bi, Sun, et al., 2024;He et al., 2024), which has provided us with the opportunity to investigate mitogenomic plant phylogeny and cytoplasmic evolution through the growing number of reported plant mitogenomes.In this study, we newly assembled two organelle genomes of S. palustris using High-Fidelity (HiFi) sequencing.Our objectives were to: (1) analyze the repeated elements and RNA-editing sites; (2) identify HGT events between plastome and mitogenome; (3) infer the phylogenetic relationships based on the two different organelle genomes and (4) explore heterogeneity associated with maternally inherited organelle.
Our results will contribute to understanding the organelle genome evolution in the threatened aquatic plant S. palustris and provide vital genomic resources and scientific support for its future conservation.

| Plant materials and sequencing
The cultivated sample of S. palustris was collected from Wuhan Botanical Garden, the Chinese Academy of Sciences in China.Total genomic DNA was isolated from young leaves using the modified CTAB method (Doyle & Doyle, 1987) for HiFi sequencing.The construction of the sequencing library followed the standard protocol of Pacific Biosciences, and HiFi sequencing was conducted on the PacBio Sequel2 platform.In total, ca. 3 Gb of HiFi reads (genome size 0.54G; https:// cvalu es.scien ce.kew.org/ ) were generated and applied for subsequent analyses.
Additionally, the online tool GeSeq (Tillich et al., 2017) was used for the initial annotation of the mitogenome and plastome of S. palustris, with Z. marina and B. umbellatus serving as references.Annotations were further manually verified and corrected using Geneious v5.6.4 (Biomatters Ltd., Auckland, New Zealand).The annotated mitogenome and plastome have been deposited into GeneBank (Table S1) and a graphic representation of the circular organelle genome map was visualized by OGDRAW (Greiner et al., 2019).

| Prediction of RNA editing sites and codon usage
To identify RNA-editing sites in two organelle genomes PCGs of S. palustris, RNA-seq data were retrieved from GenBank with the accession Nos.SRR18139151 and SRR21619434.After filtering out poor-quality reads using Fastp v0.20.0 (Chen et al., 2018) with default settings, the high-quality reads were remapped to pt-PCGs | 3 of 13 and mt-PCGs of S. palustris using BWA v.0.7.17-r1188 (Li, 2013), respectively.To enhance the coverage and depth for each locus, the two resulting BAM files were merged using SAMtools v1.20 (Li et al., 2009).RNA-editing sites were then identified and filtered using REDItools v2.0 (Picardi & Pesole, 2013), applying the following criteria: QUAL >30, depth >100×, and p < 0.1.Moreover, the identified RNA-editing sites were categorized according to their function within the mitogenome and plastome, as detailed in Tables S2 and S3, respectively.For analyzing codon preference, the software MEGA v11 (Tamura et al., 2021) was employed to calculate the relative synonymous codon usage (RSCU) for all pt-and mt-PCGs and to evaluate the codon usage bias with default settings.

| Identification of repeat elements
The SSRs were detected by using the Perl program MISA (Beier et al., 2017), with the minimum repetition numbers of mono-, di-, tri-, tetra-, penta-, and hexanucleotides 10, 5, 4, 3, 3, and 3, respectively.Additionally, the dispersed repeats, including forward, reverse, complement, and palindromic repeat sequences were identified by REPuter (Kurtz et al., 2001).The Hamming distance parameter was set to three, with a minimum repeat size requirement of 30.

| Estimation of nucleotide substitution rates
KaKs_Calculator v2.0 (Wang et al., 2010) was utilized to calculate the pairwise Ka/Ks ratios for 56 pt-and 21 mt-PCGs by analyzing the multiple nucleotide alignments of each gene pair.Additionally, the significance of the differences in the rates of synonymous mutations between the pt-and mt-PCGs of S. palustris was evaluated using a t-test in R v3.6.3.

| Analysis of horizontal gene transfer (HGT)
To identify DNA fragments with potential horizontal transfer between the mitochondrial and plastid genomes in S. palustris, we employed the BLASTN program (Camacho et al., 2009) to search for homologous sequences across the two organelle genomes with an E-value of 1e-5 and a percent identity threshold of 80%.Then, all HiFi reads were remapped to organelle genomes using Minimap2 v2.28 (Li, 2021) with recommended settings.The boundaries of each transferred fragment sequence were then manually checked using IGV software (Thorvaldsdóttir et al., 2013).Additionally, we counted the 500 bp regions before and after the ends of the transfer fragment boundary.
The conserved alignment of pt-and mt-PCGs was concatenated and used to construct the phylogenetic tree based on the maximum likelihood method implemented in IQ-Tree v2.1.4(Minh et al., 2020) following 1000 bootstrap replicates performed for each dataset.

| Relative synonymous codon usage analysis
Codon usage frequencies for the 30 unique mt-PCGs and 79 pt-PCGs were estimated and RSCU values for each codon were presented in Figure 2. In the mitogenome of S. palustris, CUU (Leu) exhibited the highest RSCU value of 1.53, followed by UGA (End) at 1.39.In contrast, the codons with the lower RSCU value included CUA (Leu) and AUA (Ile) at 0.66.Unlike in the mitogenome, the plastome of S. palustris, UUA (Leu) showed the highest RSCU value for UUA (Leu) at 1.97, with the lowest recorded for AGC (Ser) at 0.31.

| RNA editing and repeat elements analyses
RNA editing is crucial for post-transcriptional modifications within plant organelle genomes.Based on the transcriptome evidence, 296 RNA editing sites were identified across the 30 mt-PCGs of S. palustris (Figure 3a).Among these, nad7 had the highest number of RNA editing sites (61), while ccmFC, nad2, and nad9 contained only one.Notably, 12 distinct types of RNA editing were identified in S. palustris mitogenome and predominantly characterized by C-U conversions, which accounted for 86% (254 sites) of the edits, while G-A took possession of 9% (27 sites).Similarly, in the plastome of S. palustris, 142 RNA editing sites were predicted, with psaA showing the highest frequency of RNA editing events (28 sites), and over half of these sites (65%) involved C-U conversions (Figure 3b).
Analysis of repeat elements in the S. palustris mitogenome revealed a total of 92 simple sequence repeats (SSRs) and 480 dispersed repeats with lengths of at least 30 bp (Figure 4a; Figure 4c).
In contrast, the plastome contained only 66 SSRs and 61 dispersed repeats.Among these, tetranucleotide SSRs were the most prevalent in the mitochondrial genome, constituting 31 (33.69%) of the total, whereas mononucleotide SSRs dominated in the chloroplast genome, representing 36 (54.54%) of the total.Regarding dispersed repeats, 231 were forward (F) and 249 were palindromic (P) in the mitochondrial genome.Notably, no reverse (R) or complementary (C) repeats were found, as shown in Figure 4a.The plastome contained 22 forward repeats, 32 palindromic repeats, four reverse repeats, and three complementary repeats, with the most common repeat lengths ranging from 30 to 39 bp (Figure 4b).

| The substitution rates of mt-and pt-PCGs
We assessed the variation in synonymous mutation rates (Ks) within the mitogenome and plastome of S. palustris (Figure 6a,c).Our findings revealed a significant difference in the Ks ratios between the two organelles, with average values of 0.136 for mt-PCGs and 0.392 for pt-PCGs (Figure 6d).Moreover, comparing the pairwise Ka/Ks ratios among shared mt-and pt-PCGs of S. palustris with those of 13 other monocots, we observed that most of the PCGs exhibited Ka/Ks ratios below 1, suggesting strong purifying selection during the evolution history (Figure 6b,c).Notably, the genes, including atp4, ccmB, nad3, and sdh4 from the mitogenome, along with rps7 from the plastome, displayed Ka/Ks ratios exceeding 1, implying that they might undergo positive selection.

| Phylogenetic analysis by mitogenome and plastome
To investigate the phylogenetic position of S. palustris, maximum likelihood trees were reconstructed for 26 species representing the main lineages of angiosperms using 21 mt-PCGs and 56 pt-PCGs The organelle genomes map of Scheuchzeria palustris.separately.The resulting phylogenies for all selected species were well-supported, with most nodes displaying robust bootstrap supports (BS = 100; Figure 7).However, the topologies of the plastid and mitochondrial trees exhibited slight differences, particularly within the Alismatales.The mitochondrial tree highly supported that S. palustris was sistered to Butomus umbellatus and Stratiotes aloides (BS = 95; Figure 7).In contrast, the plastid analysis indicated that S. palustris was a closer relationship between S. palustris and (P hyllospadix + Zostera + Cymodocea) (BS = 100; Figure 7).

| DISCUSS ION
Organelles in plants act as powerhouses for energy metabolism, supplying the essential capacity needed to sustain the life processes of plants (Maliga, 2022;Oldenburg & Bendich, 2015).Analyzing plant organelle genomes not only enhances our understanding of their functions, inheritance, and replication but also provides insights into their evolution and adaptation (Gutiérrez et al., 2023  The notable size discrepancy between these organelle genomes may be attributed to an abundance of repetitive sequences, specifically dispersed repeats (mt: 480 vs. pt: 61; Table S5), in the mitogenome   (Wang, Kan, et al., 2024;Wang, Liu, et al., 2024;Zhou et al., 2014).Here, the GC content of the S. palustris mitogenome was approximately 47.33%, which is comparable to that of closely related species, with about 49.10% in B. umbellatus and 47.21% in C. nodosa.However, it was higher than the GC content in the plastome of S. palustris (ca.37.31%), which may account for the observed differences in codon usage bias between PCGs of two organelle genomes (Figure 2).
RNA editing is a crucial post-transcriptional modification in spermophytes that alters the genetic information in mRNAs, which can influence multiple biological functions, including the regulation of gene expression and adaptation to environmental changes (Edera et al., 2018;Szandar et al., 2022).Recently, increasing evidence has indicated that RNA editing is widely distributed among organelle genes, likely playing a key role in regulating genetics and energy metabolism (Li et al., 2019;Yang, Fu, et al., 2022;Yang, Zhao, et al., 2022).In our study, based on RNA-seq data, we F I G U R E 5 Homology analysis of the mitogenome and plastome of S. palustris.generation.The regulation of its RNA editing may significantly influence the efficiency of respiratory metabolism (Ramadan et al., 2022).In S. palustris, the RNA editing of nad7 may contribute to its ability to survive in extreme environments and improve its adaptability to environments such as low temperature or high altitude.This editing could impact the overall growth and development of the plant by modifying the structure of the protein, enabling it to participate effectively in metabolic processes even TA B L E 1 Homologous fragments between mitogenome and plastome in Scheuchzeria palustris.under unfavorable conditions.Compared to mt-PCGs, fewer RNA editing sites (142 sites) were observed in the pt-PCGs of S. palustris, which is higher than those reported in most angiosperm plastomes (Zhang et al., 2023).Similar to previous studies (Chu & Wei, 2019;He et al., 2024), C-to-U was the predominant type of editing observed in both mt-(86%) and pt-PCGs (65%) of S. palustris, potentially linked to adaptation to environmental conditions, such as cold temperatures and high altitudes.Moreover, the relatively high frequency of RNA editing in pt-PCGs suggested that the regulation of photosynthesis-related genes may play a more critical role in the adaptation and survival of S. palustris compared to genes involved in mitochondrial respiration.Additionally, genes undergoing positive selection are generally regarded as adaptations of plants to environmental stress during the evolution of plant organelle genomes (Eshel et al., 2022).In comparison to 13 other monocots, most mt-and pt-PCGs undergo purifying selection (Ka/Ks < 1), however, four mt-PCGs (atp4, ccmB, nad3, and sdh4) and one pt-PCG (rps7) exhibit a high Ka/Ks ratio (>1), indicative of positive selection, which may be attributed to their adaptation to specific environmental conditions.Interestingly, sdh4 plays a significant role in cellular responses to oxidative stress (Huang & Millar, 2013) and rps7 encodes a ribosomal protein that plays a crucial role in protein synthesis within the cell (Chen et al., 2022), indicating these may assist S. palustris in adapting to specific environments.

t p A a t p B a t p E a t p F a t p H a t p I c c s A c e m A m a t K n d h A n d h B n d h C n d h D n d h E n d h F n d h G n d h I n d h J n d h K p e t A p e t B p e t G p s a A p s a B p s a C p s a I p s a J p s b
Frequent rearrangements and horizontal gene transfers within organellar genomes have been widely proven to occur and play a pivotal role in plant evolution, which can result in the loss or gain of genes and changes in genome size (Cheng et al., 2021;Xu et al., 2023).In our study, a total of 35 mitochondrial plastid DNA (MTPT) with a total length of 58,479 bp were identified in S. palustris, accounting for 13.9% of mitogenome, which is significantly higher than most other monocots, such as Apostasia shenzhenica (5.12%; Ke et al., 2023) and Eichhornia crassipes (6.74%;He et al., 2024).The abundant repeat sequences in the mitogenome of S. palustris and may explain the high proportion of MTPT observed.Here, dispersed repeats were detected near most of the MTPT (Table S4), likely facilitating frequent interorganellar recombination, which can lead to variations in genome size and gene content (Cheng et al., 2021;Li et al., 2023).In line with previous studies (He et al., 2024;Xu et al., 2023), we identified 12 intact tRNA genes in MTPT (Table 1), supporting the frequent transfer of tRNA genes from chloroplasts to mitochondria.Interestingly, we found many intact pt-PCGs ( 23) in MTPT, which may explain why pt-PCGs of S. palustris have significantly more RNA editing sites compared to most flowering plants.
Horizontal gene transfer (HGT) is a mechanism of genetic material transmission that has been identified as a significant driving force in biological evolution (Wang, Kan, et al., 2024;Wang, Liu, et al., 2024).
The mitogenome of S. palustris contains several photosynthesisrelated genes derived from the plastome, which likely enhance the photosynthetic efficiency of S. palustris and facilitate its adaptation to lower-temperature environments.Among these, psaA, which encodes for a protein subunit of photosystem I (PSI), has been proven to contribute to the adaptation of novel habitats and low temperatures (Gao et al., 2019;Song et al., 2020;Yu et al., 2023).Here, the highest RNA-editing events (28 sites) were identified in psaA in intact PCGs of MTPT, which might contribute to the adaptation of S. palustris to unique habitats, such as low-temperature and high-altitude environments.
Due to their maternally inherited, haploid nature, and ease of assembly, plant plastomes have been widely utilized to resolve evolutionary relationships across different lineages (Li et al., 2024;Mwanzia et al., 2020).In contrast, the challenges associated with obtaining plant mitogenomes have limited their use in phylogenetic studies (Guo et al., 2023;He et al., 2024).In this study, the phylogenetic relationships of the main lineages, as derived from both sets of organelle genomes, were consistent with the APG IV system and previous research (Hu et al., 2023;Li et al., 2019).Notably, in Alismatales, the placement of S. palustris showed conflict between the mitochondrial and plastid trees, which is likely due to significant differences in the substitution rates of mt-and pt-PCGs in S. palustris (Figure 6; p < .001).
Additionally, mt-PCGs, generally associated with energy production, may be under stronger selective pressures compared to pt-PCGs, potentially influencing their evolutionary rates, as supported by analyses of positive selection.However, due to the limited sampling of Alismatales (only seven species) in our study, we refrain from drawing firm conclusions until more mitogenomes from Alismatales become available.In addition, future work can include a comparative analysis of positively selected or RNA-editing genes across closely related species to explore whether similar genomic patterns are observed in other regions, which can provide deeper insights into the adaptation strategies of aquatic species.

| CON CLUS ION
In this study, we assembled the complete organelle genomes of Photosystem IIPhotosystem I

F
Analysis of RSCU of organelle genomes in S. palustris.(a) Analysis of RSCU in the S. palustris mitogenome.(b) Analysis of RSCU in the S. palustris plastome.etal., 2022).In our study, we successfully assembled the complete mitogenome and plastome of the threatened aquatic plant S. palustris.Consistent with the typical organelle genome structure in Alismatales(Chen et al., 2022;Petersen et al., 2017), both genomes are circular, with 420,724 bp and 158,573 bp in size, respectively.

(
Figure 4), which have been observed in other species, i.e., Dystaenia takeshimana (Park & Park, 2023).Although plant mitogenomes are typically large, they generally encode around 50-60 genes (Wang, Kan, et al., 2024; Wang, Liu, et al., 2024).The mitogenome F I G U R E 3 Characteristics of the RNA editing sites identified in PCGs of S. palustris organelle genome.(a) The distribution of RNA editing sites in mt-PCGs.(b) The distribution of RNA editing sites in pt-PCGs.The distribution of SSR and dispersed repeats in the S. palustris organelle genomes.(a) The types of SSR and dispersed repeats; (b) and (c) represent the number of dispersed repeats in the plastome and mitogenome of S. palustris.
editing sites across 17 mitochondrial proteincoding genes (mt-PCGs), with nad7 exhibiting the highest frequency at 61 sites, consistent with the typical range of RNA editing sites in angiosperms, approximately 300-500(Edera et al., 2018).As a key component of the mitochondrial respiratory chain complex I, nad7 primarily facilitates electron transfer and energy F I G U R E 6 Boxplots of pairwise nucleotide substitution rates of S. palustris and other thirteen monocots.(a) Ks ratios in mt-PCGs; (b) Ka/ Ks ratios in mt-PCGs; (c) Ks and Ka/Ks ratios in pt-PCGs with purple representing Ks and green representing Ka/Ks; (d) Ks ratios between the two organelles.
K p s b N p s b T r b c L r p l1 4 r p l1 6 r p l2 0 r p l2 r p l3 The phylogenetic relationships of S. palustris and 25 other species based on shared mt-and pt-PCGs.Only the bootstrap value less than 100 was shown in the node.
Scheuchzeria palustris, with sizes of 420,724 bp for the plastome and 158,573 bp for the mitogenome.Our results revealed a high abundance of RNA editing in pt-PCGs, which may be attributed to frequent horizontal gene transfer within organellar genomes, playing a key role in rapid habitat adaptation.Five PCGs from the organellar genomes were detected under positive selection, potentially contributing to its unique adaptations to low-temperature and highaltitude environments.Additionally, phylogenomic analyses based on pt-and mt-PCGs indicated conflicting positions of S. palustris within Alismatales, suggesting significant differences in the substitution rates of mt-and pt-PCGs in this species.The organelle genomes of the threatened aquatic plant S. palustris provide vital genomic resources and scientific support for its future conservation.AUTH O R CO NTR I B UTI O N SZhi-Zhong Li: Conceptualization (lead); formal analysis (equal); supervision (equal).Jin-Ming Chen: Supervision (equal).Xiang-Yan He: Formal analysis (equal); investigation (equal); writing -original draft (equal).